J o u n i a i u/ V w r o ~ h e , m n r Vol ~ , 31. pp 613 620 Pergamon Press Ltd. 1978. Printed in Great Britain
EFFECT OF THE ADMINISTRATION OF L-~-HYDROXYTRYPTOPHANA N D A MONOAMINE OXIDASE INHIBITOR O N GLUCOSE METABOLISM IN RAT BRAIN KAR-LITWONG' a n d GERTRUDE M . TYCE Department of Physiology and Biophysics, Mayo Clinic and Mayo Foundation. Rochester, M N 55901, U.S.A. (Received 14 December 1977. Accepted 9 March 1978)
Abstract-The effects of the presence of large amounts of 5-HT and of its precursor 5-HTP in brain on cerebral utilization of glucose were studied. [U-14C]Glucose was injected to fed rats that had previously been treated with L - ~ - H T PL, - ~ - H T P and an inhibitor--N-[~-(2-chlorophenoxy)-ethyl]-cyclopropylamine hydrochloride (Lilly-51641)-of MAO, or Lilly-51641 alone. Such treatment increased the concentrations of 5-HTP and 5-HT in the brain. After treatment with 5-HTP and LiIIy-51641, and to a lesser extent with Lilly-51641 alone, the concentration of glucose in plasma was increased. However. the uptake of glucose by the brain did not appear to be proportionately increased, and this suggested an impairment in this mechanism. After the administration of Lilly-51641 alone and more especially of Lilly-51641 plus 5-HTP, the concentration of glucose in the brain was increased. This increase was thought to be due to an impairment of glucose utilization, because the flux of I4C from glucose to amino acids in the brain was reduced. The concentrations of most major amino acids in the brain were not greatly affected by these treatments. GABA and alanine concentrations in the brain were modestly increased after treatment with 5-HTP alone or in combination with Lilly-51641. The present results suggest that the metabolism of glucose to amino acids in the brain is altered when the concentration of 5-HTP, or more especially that of 5-HT, in the brain is increased.
IT HAS been shown that the brain can, under certain circumstances, utilize ketone bodies (HIMWICH, 19756) or lactate or pyruvate (HIMWICH, 1 9 7 5 ~ )but that under normal conditions glucose is its major source of energy. Yet it has only very small reserves of this carbohydrate, and thus it depends o n a supply of glucose from plasma for the maintenance of normal cerebral function. Glucose is metabolized rapidly in brain in tic0 into glutamate, aspartate, glutamine, and GABA-amino acids that are in equilibrium with the tricarboxylic acid cycle intermediates. Previous reports have shown that this metabolism is slowed after hepatectomy (TYCE et ul., 1971) and after treatment with L-DOPA, whether given alone or with inhibitors of M A 0 (EC 1.4.3.4) or of L-aromatic amino acid decarboxylase (EC 4.1.1.28) (TYCE, 1971; TYCE & OWEN,1973; TYCE,1976). NICKLAS et al. (1974) have also shown a n association between the turnover of glutamate, aspartate and glutamine and increased formation of catecholamines in the brain. 5-HTP, the precursor of serotonin, has been used t o treat depression and schizophrenia (SANO, 1972; WYATTe t a / . , 1972; MATUSSEK e t a/., 1974; TAKAHASHI e t a/., 1975). However, information about the interaction between the indoleamines and the metabolism of glucose and T o whom correspondence should be addressed. used: 5-HTP, 5-hydroxytryptophan; MAOI, monoamine oxidase inhibitor. I
Abbreriations
613
amino acids in the brain is fragmentary. The present studies were undertaken in a n attempt t o elucidate the effect of 5-HTP, the immediate precursor of serotonin, alone or combined with a n MAOI, o n glucose metabolism in the brain.
MATERIALS AND METHODS Animals. Male Sprague-Dawley rats (25c390 g) were used throughout the study. Water and food were supplied ad lib. In the experiment, two cannulas were placed under ether anesthesia, one in the tail vein for purposes of infusion and injection and the other one in the femoral artery from which blood samples (0.5 ml) could be collected for measurements of glucose and radioactivity. The rats recovered very quickly from anesthesia, but experiments were delayed for 2 h after anesthesia so that the hyperglycemic effect of ether would no longer be apparent. During this 2-h postoperative period, 0.9% saline was infused via the tail vein at a rate of 1.25 ml/h. Investigation of 5-HTP and 5-HT in bruin afier rreatment with 5-HTP and Lilly-51641. L - ~ - H T P ,purchased from Sigma Co. (St. Louis, MO, U.S.A.), was used in these experiments. 5-HT (serotonin creatinine sulfate) was obtained from the Regis Chemical Co. (Chicago. IL., U.S.A.). The MA01 N-[~-(2-chlorophenoxy)-ethyl]-cyclopropylamine hydrochloride (Lilly-51641) was a generous gift from Dr. R. Fuller, Eli Lilly & Co. (Indianapolis, IN, U.S.A.). 5-HTP and Lilly-51641 were prepared in isotonic saline at a conc of 10mg/ml. Animals receiving only 5-HTP (30mg/kg, i.v.) were killed at 15, 30, 45. 60 and 120min
614
KAR-LITWONGand G. M. T\CF
after its administration. Animals receiving Lilly-51641 (10 mg, kg, i.p.) and 5-HTP (10 mg/kg, 1.v.) were injected
with the MA01 I h before the 5-HTP was given. and they were killed at 5. 15, 30, 45, 60 and 120 min after the amino acid injection. Some animals received the Lilly-51641 only, 30 min before they were killed. Control animals received injections of the vehicles. When the animals were killed, the brains were removed rapidly and frozen in pulverized dry ice. Homogenates of the brain were rapidly prepared in 0.1 N HCI, and the method used for measurement of 5-HTP and 5-HT was & ENGELMAN (1971). Briefly, that described by LOVENBERG frozen brain was homogenized in 0.1 N HCI. 5-HT was extracted from the homogenate, after adjusting to pH 10, into washed n-butanol. After three washes with pH 10 borate buffer. heptane was added to aliquots of the butanol extract, and 5-HT was then back-extracted into 0.1 N HCI. For determination of 5-HTP, the brain homogenate was extracted with peroxide-free ether to remove 5-hydroxyindoleacetic acid and then made alkaline and washed again with washed butanol twice to remove 5-HT. 5-HTP was measured in the remaining aqueous phase. The fluorescence of 5-HT and 5-HTP in separate extracts was read at 300 nm 540 nm (acti>ation,emission)in an Amino Bowman spectrophotofluorometer (American Instrument Company. Inc.. Silver Spring, MD, U.S.A.).The apparent reeovery of 5-HT was 63.99 1.00", ( n = 67). 5-HTP was recovered reproducibly by these extraction procedures. It was not possible. however, to calculate its recovery accuratelq. because the volume of the aqueous phase present in the washed solvents used for the four extractions could not be ascertained. Internal standards of 5-HT and 5-HTP were added to duplicate samples of all homogenates before analysis so that corrections could be made for aliquot tolumes and for these recoveries. Glucose und cimino acid studies. Two hours after the tail>ein cannulation, 5-HTP (30 mg/kg, i.v.) was given to the rats. The injection of 20pCi of [U-i4C]glucose (0.24 mg in 0.2 ml of 0.9')" saline) (New England Nuclear) was performed I5 min after the injection of 5-HTP. The rat was then positioned in a freeze-blowing machine. Four and one-half minutes after administration of the radioactive glucose. a blood sample (about 0.5ml) was drawn from the femoral artery. The brain tissue was obtained after a further 30 s (5 min after [U-'4C]glucose injection) by the 1974). For brain-blowing technique (VEECH& HAWKINS, the rats receiving both the MAOI and 5-HTP, LiIly-51641 (I0 nig.'kg. i.p.) was first administered after the 2-h postanesthetic period. Thirty minutes after treatment with the MAOI. 5-HTP (10 mg/kg, i.v.) was administered, and 20 pCi of labeled glucose was also given to the rats 15 rnin later. Blood and brain samples were collected at 4.5 and 5 min after [U-'4C]glucose injection as before. ilnrrl~tictrlprocedures. The preparation of plasma and brain extracts for analysis for glucose and free amino acids followed the detailed procedures of TYCE& OWEN(1973) and TYCF(1971). Plasma protein was precipitated by the addition of perchlorie acid (0.33 M), and a clear extract was obtained by centrifugation. Frozen brain was homogenixd with 3 vol. of perchloric acid (3 M). Perchlorates were remo\,ed later by adding 5 M K O H to pH 5-7. The pH of the extract was finall) adjusted to 2.2 for amino acid assa). The glucose concentrations in plasma and brain extracts were measured by the hexokinase (EC 2.7.l.l)-glucose-6-phosphate dehydrogenase (EC 1.1.1.49) method (SLFIN.1965). The concentrations of free amino acids were
determined by means of a Bcckman amino acid analyzer (TYCE,1971). The amount of protein-free brain extract put on the column was equivalent to 0.05-0.1 g of brain tissue. Deterrninarion of rudioncririrr. The radioactivity in the protein-free extracts of both blood and brain ymples was measured by counting with Insta-Gel (Packard Instrument Co., St. Louis, MO, U S A ) in a Packard liquid scintillation counter. An external standard was used for correction of the quench. The radioactivity of the glucose. lactate, and amino acids was measured by passing the emuent from the column through a flow cell. packed with anthracene, in a liquid scintillation counter (TYCE,1971). Calculations. Brain glucose values were corrected for residual blood glucose in brain (3"" of the fresh wt) as described by FLOCKer a/. (1969). The flux of 14C from glucose in plasma to metabolites in the brain was caleulated by expressing I4C in a metabolite as a function of the specific activity of the glucose in plasma and thus obtaining its plasma glucose equivalent. Similar calculations were done to obtain brain glucose equivalents and brain glutamate equivalents in metabolites. The statistical significance of the results was determined by Student's f test. Data are given as means f S.F.M. RESULTS
Changes of hehacior of the rats after M A 0 1 and 5-HTP
A single injection of 5 - H T P (30mg/kg, i.v.) produced n o abnormal behavior in the rats. However. the combined treatment of Lilly-51641 (10 mg/kg. i.p.) and 5-HTP (30 mg/kg, i.v.) resulted in ataxia, flexion, stretch of limbs and body, intensive tremors, and heavy salivation. T h e dark red color of the blood from such animals suggested a lack of oxygen. When the rats were treated with a lower dose of 5-HTP (10mg/kg, i.v.) after the Lilly-51641 (10 mg/kg, i.p.), only slight involuntary movement of the toes and light salivation were seen. This abnormal behavior occurred about S m i n after the injection of 5-HTP and returned t o normal after about 3 W 5 min. Changes of 5-HTP and 5-HT in brain T h e maximum concentrations of 5 - H T P and 5-HT were found in the brain at 15 rnin after the injection
6t
Time,
rnin
FIG. 1. Etfects of 5-HTP alone ( e - 4 ) or Lilly-51641 on concentration of 5-HTP in rat plus 5-HTP (D-M) brain. In Figs. 1 and 2, 5-HTP was given to rats at time zero and LilIy-51641 was given at 1 h before 5-HTP. Data shown are mean f S.E. of six determinations.
61 5
5-Hydroxyindoles and cerebral glucose metabolism
I
15
30
I
1
1
45
60
I20
Time,
FIG. 2. Effects of 5-HTP alonc (o---+)
min
or Lilly-51641 plus 5-HTP (m5-HT in rat brain.
~
W)
on concentration of
of 5-HTP when it was injected alonc or with the M A 0 inhibitor (Figs. 1 and 2). Consequently, studies of the effects of 5-hydroxyindoles on ['4C]glucose metabolism were done at this time. When Lilly-51641 was given alone, the concentrations of 5-HTP and 5-HT in the brain were not changed.
of threonine in the brain of rats treated with 5-HTP or with Lilly-51641 but not in the rats that received the combined injection. However, these changes were very minor and were within the range of variation found in untreated rats in this laboratory (TYCE, 1976).
Ckarig~soJ' plasrna aid brain glucose
Radioactivity in brain at 5 rnin after adriiinisrratiori of labeled glucose
5-HTP injection alonc failed to cause a significant increase in the plasma concentration of glucose in rats (Table 1). A small increase in plasma glucose levels occurred in rats receiving only Lilly-51641, but the hyperglycemia was more pronounced after the combined administration of Lilly-51641 and 5-HT (Table 1). Brain glucose was slightly, but not significantly, increased by the injection of 5-HTP alone and was significantly increased by the injection of Lilly-51641. A highly significant increase of brain glucose was found in the rats injected with Lilly-51641 plus 5-HTP (Table 1). Changes of amino acids in brain Changes in the concentrations of amino acids in the brain were not great after the treatments with Lilly-51641 and 5-HTP. Alanine and GABA were significantly increased in the brain of rats treated with 5-HTP alone or with Lilly-51641 and 5-HTP (Table 2). Small increases were found in the concentrations
Compared with the controls. 5-HTP injection caused pronounced increases in the radioactivity as glucose and reduced the amounts of radioacti\e amino acids and of lactate (Table 3). More extensive changes of these proportions were caused by Lilly-51641. When both Lilly-51641 and 5-HTP were injected into the animals, radioactive glucose was increased more than 60';/, and the radioactivity as amino acids was significantly decreased (Table 3). Treatment with 5-HTP changed slightly the specific radioactivity of glucose in the brain 5 min after the injection of labeled glucose (Table 4). The administration of Lilly-51641 also significantly reduced the specific radioactivity of glucose, and a more extensive reduction was found after the combined injection of Lilly-51641 and 5-HTP (Table 4). Similarly, specific radioactivities of the major amino acids in brain were also decreased after the injection of 5-HTP or Lilly-51641 or both (Table 4).
TABLE1. CONCENTRATION OF PLASMA
AND BRAIN GLUCOSE*
Treatment
Plasma Glc (pmol/ml)
Brain glucose @mol/g)
Control ( 6 ) 5-HTP (5) Control (6)
9.85 f 0.35 11.86 f 0.98
2.31 f 0.18
M A 0 1 (6) M A 0 1 + 5-HTP (6)
11.53 _+ 1.02t 17.26 f 1.281
8.90 f 0.42
2.02 f 0.11 1.89 f 0.15 2.54 _+ 0.22t 3.76 f 0.21:
* Figures in parentheses represent the number of animals; values are means f appropriate controls. t P < 0.0s. 1P < 0.001.
S.E.M.
against
KAR-LITWONCand G. M. TYCE
616
TABLF2. CONCENTRATION OF AMINO Substance Taurine Aspartate Threonine Glutamine Glutamate Glycine Alanine GABA
ACIIIS IN B R A I N
(pmol/g*)
Control (6)
5-HTP (5)
Control (6)
MA01 (6)
MA01 + 5-HTP (6)
4.68 f 0.25 3.08 f 0.17 0.47 f 0.02 6.38 f 0.22 11.77 f 0.42 0.62 k 0.02 0.32 f 0.01 1.45 f 0.05
4.19 f 0.47 3.11 f 0.16 0.54 k 0.02t 6.76 k 0.15 11.78 f 0.14 0.67 f 0.03 0.35 f 0.0lt 1.73 k 0.081
4.98 f 0.31 3.14 f 0.12 0.49 f 0.02 6.36 f 0.30 11.32 f 0.21 0.63 f 0.03 0.34 5 0.01 1.61 f 0.11
4.60 f 0.22 3.29 f 0.09 0.56 f 0.02t 6.67 f 0.17 11.42 f 0.33 0.60 f 0.02 0.34 f 0.01 1.63 f 0.10
4.89 f 0.09 3.26 f 0.10 0.54 f 0.02 7.10 f 0.28 11.00 f 0.17 0.70 f 0.04 0.43 f 0.02: 1.99 f O.12t
* Figures in parentheses represent number of animals; values are means f t P < 0.05.
against appropriate controls
S.F.M.
:P < 0.01.
TABLF3. RADIOACTIVITY I N BRAIN 5 min Substance Glucose Lactate Aspartate Glutamine Glutamate Alanine GA BA
AFTER INJECTION or L A B ~ L E DGLUCOSE*
Control (6)
5-HTP (5)
Control (6)
MA01 (6)
MA01 + 5-HTP (6)
42.75 f 2.82 8.31 f 0.79 5.26 f 0.25 4.84 f 0.39 24.48 f 1.40 1.44 k 0.14 2.12 f 0.18
50.63 f 2.54 7.86 _+ 0.68 3.74 f 0.35t 4.06 f 0.32 20.19 f 1.198 1.29 f 0.12 1.68 & 0.16
41.92 f 2.04 7.76 f 0.40 5.34 f 0.04 4.80 f 0.21 24.25 f 1.29 1.51 f 0.10 2.04 f 0.15
53.32 f 3.44t 6.42 f 0.465 3.89 f 0.38t 3.06 f 0.39t 18.33 f 1.665 1.14 f 0.18 1.90 f 0.29
68.24 f 2.12: 6.08 f 0.905 2.02 f 0.13: 2.04 f 0.19: 10.74 f 0.68: 0.78 f 0.13t 1.03 f 0.17:
* Figures in parentheses represent the number of animals; data are expressed as percent of total 14C in brain; values are means f S.E.M. against appropriate controls. t P < 0.01.
:P < 0.001.
$P
< 0.05.
RADIOACTIVITY TABLE4. SPECIFIC
Substance Glucose Aspartate Glutamine Glutamate Alanine GABA
OF G L U C O S ~AND AMINO ACIDS I N RAT BRAIN
(d.p.m./pmol x lo-’)*
Control (6)
5-HTP (5)
Control (6)
MA01 (6)
MA01 + 5-HTP (6)
65.83 f 7.49 5.31 k 0.56 2.37 f 0.36 6.45 f 0.79 14.31 f 2.07 4.51 f 0.61
52.50 f 2.28 2.87 f 0.263 1.42 f 0.Oq 4.06 f 0.18t 8.69 k 0.53t 2.34 f 0.29t
59.46 f 5.25 4.70 f 0.27 2.07 k 0.08 5.91 f 0.40 12.03 f 1.08 3.64 f 0.53
45.26 f 2.861. 2.55 & 0.316 1.00 f 0.165 3.45 f 0.393 7.07 f 1.21t 2.51 f 0.45
37.34 & 2.131 1.29 & 0.145 0.60 f 0.076 2.00 f 0.176 3.78 f 0.66s 1.02 f 0.11s:
* Figures in parentheses represent the number of animals; values are means f t P < 0.05.
S.E.M.
against appropriate controls.
:P < 0.01.
5 P < 0.001.
Incorporation of labeled glucose into its nietabolites in brain
The administration of Lilly-51641 alone or with 5-HTP reduced the total 14C in brain 5 min after the intravenous injection of labeled glucose (Table 5). However, the plasma glucose equivalent in brain was unchanged after treatment with Lilly-51641, with 5-HTP, or with Lilly-51641 plus 5-HTP. This suggested that the uptake of glucose into the brain from the circulation was not increased after these treatments, even though the plasma concentration of glucose was increased (Table 1). The flux of 14C from plasma glucose to lactate and alanine was not appreciably affected by the adminis-
tration of Lilly-51641 or 5-HTP or both (Table 5). O n the other hand, the flux of 14C to the major amino acids other than alanine in the brain was sharply decreased after injection of 5-HTP or Lilly-51641, and the decrease was more pronounced after combined treatment with Lilly-51641 and 5-HTP (Table 5). Equivalents of glucose and glutarnate in their rnetaholites
The brain glucose equivalents in glutamate and in aspartate in the brain were reduced after injection of 5-HTP (Table 6). The injection of Lilly-51641 alone also caused a reduction of brain glucose equivalents
5-Hydroxyindoles and cerebral glucose metabolism TABLE 5. FLUXOF I4C FROM
PLASMA GLUCOSE TO METABOLITES IN BRAIN
Measurement
5 min
617
AFTER THE INJECTION OF LABELED
GLUCOSE*
Control (6)
5-HTP (5)
Control (6)
MA01 (6)
MA01 + 5-HTP (6)
57.63 f 0.35
47.86 f 1.717
56.54 f 3.24
41.66 f 3.83t
36.89 f 1.703
1. Specific activity of
plasma glucose (d.p.m./pnol x 2. Total I4C in brain (d.p.m./g x 3. Plasma glucose equivalent in brain 4. For lactate and alanine 0,; of total Plasma glucose equivalent 5. For amino acids except alanine :,;of total l4C Plasma glucose equivalent
310.23 f 32.81
237.88 f 6.46
275.36 f 10.69 212.56 f 8.293
204.27 f 10.873
5.33 f 0.36
5.00 f 0.23
4.92 i. 0.23
5.24 f 0.30
5.55 f 0.22
9.75 f 0.89 0.52 f 0.07
9.15 f 0.61 0.46 f 0.03
9.27 f 0.43 0.45 f 0.02
7.56 f 0.55 0.39 f 0.03
6.86 f 1.03 0.37 f 0.04
36.70 f 2.06 1.95 f 0.16
29.67 f 1.88 1.47 f 0.06t
36.34 f 1.72 1.78 f 0.06
27.19 f 2.36 1.39 0.06:
15.82 f 1.01 0.87 f 0.041
* Figures in parentheses represent the number of animals; values are means f
S.E.M.
against appropriate controls.
t P < 0.05. 1 P < 0.001
TABLE6. BRAINGLUCOSE
Metabolites Lactate Aspartate Glutamine Glutamate Alanine GABA
EQUIVALENTS I N GLUCOSE METABOLITES I N BRAIN
Control ( 6 )
5-HTP (5)
Control (6)
MA01 (6)
0.39 f 0.04 0.25 f 0.01 0.23 f 0.02 1.16 f 0.08 0.07 f 0.007 0.10 f 0.01
0.36 f 0.04 0.17 f 0.0lt 0.18 f 0.02 0.92 f 0.06$ 0.06 f 0.003 0.08 f 0.005
0.37 f 0.03 0.25 f 0.01 0.23 f 0.01 1.14 f 0.05 0.07 f 0.003 0.10 f 0.01
0.30 f 0.01 0.18 f 0.02t 0.14 f 0.02t 0.86 f 0.07t 0.05 f 0.01 0.09 f 0.01
* Figures in parentheses represent the number
(jtmol/g)* MA01 + 5-HTP (6) 0.33 f 0.04 0.11 f 0.01: 0.11 f 0.01: 0.59 f 0.03: 0.04 f 0.006t 0.055 f 0.008t
of animals; values are means f S.E.M.against appropriate controls.
t P < 0.01. $ P < 0.001. $ P < 0.05.
TABLE7. GLUTAMATE EQUIVALENTS I N
Metabolites
METABOLITES I N BRAIN
(pmol/g)*
Control (6)
5-HTP (5)
Control (6)
MA01 (6)
MA01 + 5-HTP (6)
2.30 f 0.10 2.56 0.18 0.69 f 0.45 1.01 0.04
2.36 f 0.07 2.17 f 0.15 0.76 f 0.07 0.98 f 0.08
2.25 f 0.10 2.50 0.03 0.70 f 0.03 0.96 f 0.06
1.88 0.12t 2.43 f 0.15 0.71 f 0.09 1.21 2 0.20
2.10 f 0.20 2.07 f 0.08t 0.81 f 0.13 1.04 0.13
~
Glutamine Aspartate Alanine GABA
* Figures in parentheses represent the number of animals; values are means f
S.E.M.
against appropriate controls.
t P < 0.05.
in aspartate, glutamine, and glutamate. The administration of 5-HTP and the MAOX significantly decreased the brain glucose equivalent in all major amino acids (Table 6). However, the glucose equivalent in lactate in the brain was not affected by any of the above treatments (Table 6). The brain glutamate equivalents in its metabolites were little influenced by the injection of either 5-HTP or Lilly-51641 or by combined administration (Table 7). The only significant changes from normal were a reduction of glutamine and aspartate after administration of Lilly-51641 and Lilly-51641 plus 5-HTP, respectively.
DISCUSSION Intravenous injection of 5-HTP in our experiments resulted in about a 3-fold higher level of 5-HT in the brain than in controls, and at the same time the level of 5-HTP was also increased. These results coincide with previous findings, for levels of 5-HT in the brain or in peripheral tissue have long been noted to be increased after the administration of its precursors tryptophan or 5-HTP (UDENFRIEND ef al., 1957; KNOTT& CURZON, 1974). JOHNSTON (1968) suggested the presence of two types of M A 0 in rat brain (types A and B), and sero-
618
WONG and G. M. Tvct KAR-LIT
tonin was shown to be metabolized by the type A the plasma glucose equivalent is not increased in enzyme (FULLER,1972; NEFFet al., 1974). Although these experiments under conditions in which concenconvincing proof of the existence of several forms of trations of glucose in plasma were increased, it seems M A 0 has not yet been obtained (JAIN,1977), the in- probable that uptake of glucose by the brain was in hibitor LiIIy-51641 has been shown to be selective some way impaired by treatment with 5-HTP and in its effect of blocking the metabolism of 5-HT by with Lilly-51641. M A 0 (FULLER,1972). The present treatment with Generally, injections of Lilly-51641 and 5-HTP, Lilly-51641 plus 5-HTP resulted in an approx 4-fold singly or together, did not change the amino acid higher level of brain 5-HT than that in the intact concentrations in the brain in the present studies. rats. 5-HTP was also increased after the combined 5-HTP caused a modest increase in the levels of administration of Lilly-51641 and 5-HTP. The glu- alanine and GABA. Alanine and GABA levels were cose metabolism under the influence of the MA01 also increased by 5-HTP injection after the and of 5-HTP was investigated at times when 5-HT Lilly-51641 treatment, but Lilly-51641 given alone did or 5-HTP was at its highest level after the treatment. not alter their levels. In the present experiments, administration of Changes in 5-HT levels in brain have been shown to be associated with pronounced behavioral changes 5-HTP and of Lilly-51641 reduced the conversion of in animals. Tremors have also been shown to occur glucose to GABA and other major amino acids in et al., 1971; the brain. This reduced metabolism of glucose was in mice by other investigators (MANNISTO MODIGH,1974),and it is suggested that these manifes- probably in part the reason for its accumulation in tations were due to large amounts of 5-HT acting brain. However, it is of interest that although the flux on central 5-HT receptors (ALGERI& CERLETTI, 1974; to GABA from glucose was decreased, the concenJACOBY et al., 1976). tration of GABA increased both after 5-HTP treatBoth hypoglycemic and hyperglycemic effects have ment and after the combined treatment. It has been been reported previously after the administration of suggested (POPOV& MATTHIES, 1969) that MA01 can 5-HTP or 5-HT to animals. Variations in the glyce- increase the GABA level by an inhibition of GABA mic response to these agents may be related to transaminase (EC 2.6.1.19), but in our experiments the species; thus, dogs (SIREKet al., 1966) and rabbits administration of the MA01 alone did not signifiet a[.,1952) usually show hyperglycemic re- cantly affect the concentration of GABA. (CORRELL sponses, mice demonstrate hypoglycemia (DARWISH 5-HT can produce major changes in the vasculature & FURMAN, 1975), and rats both hyperglycemia (COR- (SWANK& HISSEN,1964; DESHMUKH & HARPER, RELL et a/., 1952; LEVINE et a/.,1964) and hypoglyce- 1973: RAPELA& MARTIN,1975; MENDELOW et al., mia (MIRSKY et al.. 1957; KOBAYASHI et al., 1960). 19771, and after injection of 5-HTP, increased The responses to 5-HTP and 5-HT in rats may amounts of 5-HT can be expected in the plasma et al., UDENFRIEND depend on the strain; thus, Wistar (KOBAYASHI et al., 1957). Thus, part of the decreased 1960) and Carworth (MIRSKY el al., 1957) rats usually flux of I4C from glucose in the plasma to its metaboet lites in the brain observed after the injections of show hypoglycemia, and Sprague-Dawley (LEVINE a/., 1964) and albino (CORRELL et a/., 1952) rats 5-HTP (Table 5) could be attributed to a decrease demonstrate hyperglycemia. Another factor in rats is in cerebral blood flow (DESHMUKH & HARPER,1973) undoubtedly the state of nutrition of the animals; the induced by 5-HT present in the plasma. However, hypoglycemic response usually occurs in fasted ani- the conversion of I4C from brain glucose to metabomals (MIRSKYet a!., 1957; KOBAYASHI et al., 1960). lites in the brain was also inhibited and to a similar et a!. (1971) have also shown that extent by the 5-HTP treatments (Table 6). Because LUNDQUIST another MAOI, N-methyl-N-2-propynylbenzylaminethe glucose in the brain had been corrected for glu(pargyline), can cause hypoglycemia in mice when cose in the residual blood in the brain (although not given alone or in combination with 5-HTP. On the for glucose in the cerebrospinal flaid), this decreased other hand, the injection of 5-HT and N-benzyl- conversion of brain glucose to its metabolites suggests p-(isonicotinoy1hydrazino)propionamide (nialamide) that there is. in fact. an inhibition of glucose metabolresulted in hyperglycemia in mice (DARWISH & FUR- ism in the brain after the injections of 5-HTP, MAN, 1974). However, their effects may be mediated although an effect on cerebral blood flow cannot be by changes in the concentrations of other amines, excluded. such as tyramine (FULLER,1972), rather than 5-HT. Although the flux of 14C from glucose to the amino Glucose passes the blood-brain barrier by a special acids in brain was reduced by treatment with 5-HTP transport mechanism (BETZet al., 1976), and the in- or with Lilly-51641, the flux to lactate and alanine flux of glucose from the circulation to the brain is appeared to be unaffected. This suggests that a point influenced and regulated by the concentration of glu- of inhibition was at the entry of pyruvic acid into cose in plasma (PRATT,1976). Although the plasma the tricarboxylic acid cycle. A similar decreased flux of carbon from glucose glucose equivalent, as calculated in these experiments, is not a precise measurement of glucose uptake by to amino acids in the brain was observed previously the brain from plasma (TYCE, 1971)-some 14C is lost after DOPA administration (TYCE& OWEN,1973; as C0,-it does provide an index of uptake. Since TYCE,1976) and after phenylalanine or tryptophan
5-Hydroxyindoles and cerebral glucose metabolism administration (BARBATO & BARBATO,1969). This suggests a common effect of aromatic amino acids on glucose metabolism in brain. This effect would be caused by a competition by the aromatic amino acids for cofactors, especially pyridoxal-5-phosphate, that are necessary for the formation of glutamate, glutamine, aspartate, and GABA from tricarboxylic acid cycle intermediates. Pyridoxal-5-phosphate is also a cofactor for the enzyme L-aromatic amino acid decarboxylase and for tyrosine aminotransferase (EC 2.6.1.5) (NICKLAS& BERL,1973). It would be of interest to determine whether the metabolism of glucose is inhibited to a similar extent in rats fed with pyridoxine-deficient diets or by the administration of drugs that bind pyridoxal phosphate (for example, penicillamine and isoniazid). These problems are currently being investigated in our laboratory. However, in the present experiments, the inhibition of flux of carbon from labeled glucose to amino acids was greatest in rats in which the amine (rats treated with LilIy-51641 plus 5-HTP) rather than the amino acid precursor (rats treated with 5-HTP) was increased. Further experiments are needed to determine whether the intraventricular injection of the amines 5-HT and dopamine would elicit similar inhibition. It would also be important to ascertain what effect the injection of an inhibitor of central and peripheral decarboxylatjon (for example, 4-bromo-3-hydroxybenzyloxyamine. NSD 1055) has on glucose utilization of the brain. Compared with previous reports from our laboratory (TYCE, 1976) and from other institutions (SHIMADA er al., 1970) in which liquid nitrogen was used to kill rats or mice, the use of the brain-blowing technique to fix brain tissue showed a higher percentage of radioactivity as glucose and amino acids and a markedly lower amount as lactate. These data undoubtedly reflect a minimizing of postmortem artifacts when the more rapid method of killing was used. AcknowledgementsThe authors are greatly indebted to Mrs. JAUNEITA OGG and Mr. CURTIS GRABAUfor their skillful technical assistance.
619
DESHM~JKH V. D. & HARPERA. M. (1973) Acra ncurol. s c a d . 49, 649-658. FLOCK E. V., TYCEG. M. & OWEN C. A.. JR. (1969) Mayo Clin. Proc. 44, 387-405. FULLER R. W. (1972) Adv. Biochem. Psychophartnac. 5, 339-354. HIMWICHH. E. (1975a) Brain Metabolisni and Cerebral Disorders. 2nd ed., pp. 11-32. Spectrum. New York. HIMWICH H. E. (197%) Brain Metabolism and Cerebral Disorders. 2nd ed., pp. 33-63. Spectrum, New York. JACOBY J. H., H o w R. A., LEVINM. S. & WURTMAN R. J. (I 976) Neurophannacology IS, 529-534. JAINM. (1977) LiJe Sci. 20, 19251933. JOHNSTON J. P. (1968) Biochern. Pharrnac. 17, 1285-1297. KNOTT P. J. & CURZONG. (1974) J. Neurochem. 22, 1 0 6 5I07 1. KOBAYASHI B., UI M. & WARASHINA Y. (1960) E,docrinol. j a p . 7, 225-238. LEVINE R. A., PESCHL. A., KLATSKIN G. & GIARMAN N. J. (1964) J . d i n . Inoest. 43, 797-809. LOVENBERG S. & ENGELMAN K. (1971) in Analysis gf Biogenic Aniines and Their Related Enzymes (Methods of Biochetnical Ana/ysis, Supplemental Volunie) ( G I . I ~ D.. K ed.) pp. 1-34. Interscience Publishers. New York. LUNDQUIST I., EKHOLM R. & ERICSON L. E. (1971) Diabefologia 7, 414422. MANNISTOP., NIKKIP. & RISSANEN A. (1971) Acra pharmac. f o x . 29, 441448. MATUSSEK N., ANGSTJ., BENKERT 0..GMURM., PAPOUSEK M., RUTHERE. & WOGGONB. (1974) Adr. Biochem. Psychopharmac. 1 1, 399404. MENDELOW A. D., EIDELMAN B. H., MCCALDEN T. A. & ROSENDORFF C. (1977) Stroke 8, 322-325. MIRSKYI. A., PERISUTTI G. & JINKSR. (1957) €tidoerinology 60, 3 18-324. MODIGH K. (1974) Adr. Biochem. Psychopharmac. 10, 21 3-217. NEFFN. H., YANGH.-Y. T., GORIDIS C. & BIALEK D. (1974) Ad{,. Biochem. Psychopharmac. 11, 51-58. NICKLAS W. J. & BERLS. (1973) Brain Res. 61, 343-356. NICKLAS W. J., BERLS. & CLARKE D. D. (1974) J. Neurochern. 23, 149-157. POPOVN. & MATTHIESH. (1969) J. Neurocheni. 16, 899-907. PRATT 0. E. (1976) in Transport Phenomena in the Nerrous System: Physiological and Pathological Aspecrs (LEVIG., BATTISTIN L. & LAJTHAA,, eds.) pp. 55-75. Plenum
Press, New York. REFERENCES
RAPELAC. E. & MARTINJ. B. (1975) in Blood Flow and Metabolism in the Brain (HARPER M., JENNETTB., MILLERD. & ROWANJ., eds.) pp. 4.54.9. Churchill Liv-
ingstone, Edinburgh. ALGERIS. & CERLETTI C. (1974) Adr. Biochetn. Psychopharmar. 10, 257-259. BARBATO L. M. & BARBATO I. M. (1969) Brain Res. 13, 569578. BETZA. L., GILROE D. D. & DREWESL. R. (1976) in Transport Phenomena in the Nervous System: Physiological and Pathological Aspects (LEVIG., BATTISTINL. & LAJTHA A,, eds.) pp. 13F149. Plenum Press, New York. S. & VANDERPOEL J. C. CORRELL J. T., LYTHL. F., LONG (1952) Ant. J . Physiol. 169, 537-544. DARWISH S. A. E. & FURMAN B. L. (1974) Experrentia 30, 1306-1 307. DARWISHS. A. E. & FURMAN B. L. (1975) Br. J . Phurmac. 54, 263P.
SANOI. (1972) Folia Psychiat. neurol. jap. 26, 7-17. SHIMADA M., IWATAK., INOUES., OTOMOS. & KIHARA T. (1970) Acta Anat. Nippon 45, 149-157. SIREKA,, GEERLINC E. & SIREK0.V. (1966) Am. J , Phjsiol. 211, 1018-1020. SLEINM. W. (1965) in Methods of Enzymatic Analysis (BERGMEYER H.-H., ed.) pp. 117-123. Academic Press. New York. SWANKR. L. & HISSENW. (1964) Archs. Neurol. Chicago 10, 468472. TAKAHASHI S., KONDOH. & KATON. (1975) J. Psychjar. Res. 12, 177-187. T Y C EG. M. (1971) Biochem. Pharmac. 20, 2371-2384. TYCEG. M. (1976) J. Neurochem. 27, 1397-1403.
620
KAR-LITWONGand G. M. TYCE
E. V. & OWENC. A., JR. (1971) Expl. TYCEG. M., FLOCK bid. Med. 4, 92-103. TYCEG. M. & OWENC. A,, JR. (1973) J . Neurochem. 20, 1563-1573. UDENFRIEND S., WEISSBACH H. & BOCDANSKI D. F. (1957) J . biol. Chem. 224. 803-810.
VEECHR . L. & HAWKINS R. A. (1974) in Research Methods N. & RODNIGHTR., eds.) Vol. in Neurochemistry (MARKS 2, pp. 171-182. Plenum Press, New York. WYATTR. J., VAUGHANT., GALANTER M., KAPLANJ. & GREEN R. (1972) Science, N.Y. 177, 1124-1126.
EFFECT OF THE ADMINISTRATION OF L-~-HYDROXYTRYPTOPHANA N D A MONOAMINE OXIDASE INHIBITOR O N GLUCOSE METABOLISM IN RAT BRAIN KAR-LITWONG' a n d GERTRUDE M . TYCE Department of Physiology and Biophysics, Mayo Clinic and Mayo Foundation. Rochester, M N 55901, U.S.A. (Received 14 December 1977. Accepted 9 March 1978)
Abstract-The effects of the presence of large amounts of 5-HT and of its precursor 5-HTP in brain on cerebral utilization of glucose were studied. [U-14C]Glucose was injected to fed rats that had previously been treated with L - ~ - H T PL, - ~ - H T P and an inhibitor--N-[~-(2-chlorophenoxy)-ethyl]-cyclopropylamine hydrochloride (Lilly-51641)-of MAO, or Lilly-51641 alone. Such treatment increased the concentrations of 5-HTP and 5-HT in the brain. After treatment with 5-HTP and LiIIy-51641, and to a lesser extent with Lilly-51641 alone, the concentration of glucose in plasma was increased. However. the uptake of glucose by the brain did not appear to be proportionately increased, and this suggested an impairment in this mechanism. After the administration of Lilly-51641 alone and more especially of Lilly-51641 plus 5-HTP, the concentration of glucose in the brain was increased. This increase was thought to be due to an impairment of glucose utilization, because the flux of I4C from glucose to amino acids in the brain was reduced. The concentrations of most major amino acids in the brain were not greatly affected by these treatments. GABA and alanine concentrations in the brain were modestly increased after treatment with 5-HTP alone or in combination with Lilly-51641. The present results suggest that the metabolism of glucose to amino acids in the brain is altered when the concentration of 5-HTP, or more especially that of 5-HT, in the brain is increased.
IT HAS been shown that the brain can, under certain circumstances, utilize ketone bodies (HIMWICH, 19756) or lactate or pyruvate (HIMWICH, 1 9 7 5 ~ )but that under normal conditions glucose is its major source of energy. Yet it has only very small reserves of this carbohydrate, and thus it depends o n a supply of glucose from plasma for the maintenance of normal cerebral function. Glucose is metabolized rapidly in brain in tic0 into glutamate, aspartate, glutamine, and GABA-amino acids that are in equilibrium with the tricarboxylic acid cycle intermediates. Previous reports have shown that this metabolism is slowed after hepatectomy (TYCE et ul., 1971) and after treatment with L-DOPA, whether given alone or with inhibitors of M A 0 (EC 1.4.3.4) or of L-aromatic amino acid decarboxylase (EC 4.1.1.28) (TYCE, 1971; TYCE & OWEN,1973; TYCE,1976). NICKLAS et al. (1974) have also shown a n association between the turnover of glutamate, aspartate and glutamine and increased formation of catecholamines in the brain. 5-HTP, the precursor of serotonin, has been used t o treat depression and schizophrenia (SANO, 1972; WYATTe t a / . , 1972; MATUSSEK e t a/., 1974; TAKAHASHI e t a/., 1975). However, information about the interaction between the indoleamines and the metabolism of glucose and T o whom correspondence should be addressed. used: 5-HTP, 5-hydroxytryptophan; MAOI, monoamine oxidase inhibitor. I
Abbreriations
613
amino acids in the brain is fragmentary. The present studies were undertaken in a n attempt t o elucidate the effect of 5-HTP, the immediate precursor of serotonin, alone or combined with a n MAOI, o n glucose metabolism in the brain.
MATERIALS AND METHODS Animals. Male Sprague-Dawley rats (25c390 g) were used throughout the study. Water and food were supplied ad lib. In the experiment, two cannulas were placed under ether anesthesia, one in the tail vein for purposes of infusion and injection and the other one in the femoral artery from which blood samples (0.5 ml) could be collected for measurements of glucose and radioactivity. The rats recovered very quickly from anesthesia, but experiments were delayed for 2 h after anesthesia so that the hyperglycemic effect of ether would no longer be apparent. During this 2-h postoperative period, 0.9% saline was infused via the tail vein at a rate of 1.25 ml/h. Investigation of 5-HTP and 5-HT in bruin afier rreatment with 5-HTP and Lilly-51641. L - ~ - H T P ,purchased from Sigma Co. (St. Louis, MO, U.S.A.), was used in these experiments. 5-HT (serotonin creatinine sulfate) was obtained from the Regis Chemical Co. (Chicago. IL., U.S.A.). The MA01 N-[~-(2-chlorophenoxy)-ethyl]-cyclopropylamine hydrochloride (Lilly-51641) was a generous gift from Dr. R. Fuller, Eli Lilly & Co. (Indianapolis, IN, U.S.A.). 5-HTP and Lilly-51641 were prepared in isotonic saline at a conc of 10mg/ml. Animals receiving only 5-HTP (30mg/kg, i.v.) were killed at 15, 30, 45. 60 and 120min
614
KAR-LITWONGand G. M. T\CF
after its administration. Animals receiving Lilly-51641 (10 mg, kg, i.p.) and 5-HTP (10 mg/kg, 1.v.) were injected
with the MA01 I h before the 5-HTP was given. and they were killed at 5. 15, 30, 45, 60 and 120 min after the amino acid injection. Some animals received the Lilly-51641 only, 30 min before they were killed. Control animals received injections of the vehicles. When the animals were killed, the brains were removed rapidly and frozen in pulverized dry ice. Homogenates of the brain were rapidly prepared in 0.1 N HCI, and the method used for measurement of 5-HTP and 5-HT was & ENGELMAN (1971). Briefly, that described by LOVENBERG frozen brain was homogenized in 0.1 N HCI. 5-HT was extracted from the homogenate, after adjusting to pH 10, into washed n-butanol. After three washes with pH 10 borate buffer. heptane was added to aliquots of the butanol extract, and 5-HT was then back-extracted into 0.1 N HCI. For determination of 5-HTP, the brain homogenate was extracted with peroxide-free ether to remove 5-hydroxyindoleacetic acid and then made alkaline and washed again with washed butanol twice to remove 5-HT. 5-HTP was measured in the remaining aqueous phase. The fluorescence of 5-HT and 5-HTP in separate extracts was read at 300 nm 540 nm (acti>ation,emission)in an Amino Bowman spectrophotofluorometer (American Instrument Company. Inc.. Silver Spring, MD, U.S.A.).The apparent reeovery of 5-HT was 63.99 1.00", ( n = 67). 5-HTP was recovered reproducibly by these extraction procedures. It was not possible. however, to calculate its recovery accuratelq. because the volume of the aqueous phase present in the washed solvents used for the four extractions could not be ascertained. Internal standards of 5-HT and 5-HTP were added to duplicate samples of all homogenates before analysis so that corrections could be made for aliquot tolumes and for these recoveries. Glucose und cimino acid studies. Two hours after the tail>ein cannulation, 5-HTP (30 mg/kg, i.v.) was given to the rats. The injection of 20pCi of [U-i4C]glucose (0.24 mg in 0.2 ml of 0.9')" saline) (New England Nuclear) was performed I5 min after the injection of 5-HTP. The rat was then positioned in a freeze-blowing machine. Four and one-half minutes after administration of the radioactive glucose. a blood sample (about 0.5ml) was drawn from the femoral artery. The brain tissue was obtained after a further 30 s (5 min after [U-'4C]glucose injection) by the 1974). For brain-blowing technique (VEECH& HAWKINS, the rats receiving both the MAOI and 5-HTP, LiIly-51641 (I0 nig.'kg. i.p.) was first administered after the 2-h postanesthetic period. Thirty minutes after treatment with the MAOI. 5-HTP (10 mg/kg, i.v.) was administered, and 20 pCi of labeled glucose was also given to the rats 15 rnin later. Blood and brain samples were collected at 4.5 and 5 min after [U-'4C]glucose injection as before. ilnrrl~tictrlprocedures. The preparation of plasma and brain extracts for analysis for glucose and free amino acids followed the detailed procedures of TYCE& OWEN(1973) and TYCF(1971). Plasma protein was precipitated by the addition of perchlorie acid (0.33 M), and a clear extract was obtained by centrifugation. Frozen brain was homogenixd with 3 vol. of perchloric acid (3 M). Perchlorates were remo\,ed later by adding 5 M K O H to pH 5-7. The pH of the extract was finall) adjusted to 2.2 for amino acid assa). The glucose concentrations in plasma and brain extracts were measured by the hexokinase (EC 2.7.l.l)-glucose-6-phosphate dehydrogenase (EC 1.1.1.49) method (SLFIN.1965). The concentrations of free amino acids were
determined by means of a Bcckman amino acid analyzer (TYCE,1971). The amount of protein-free brain extract put on the column was equivalent to 0.05-0.1 g of brain tissue. Deterrninarion of rudioncririrr. The radioactivity in the protein-free extracts of both blood and brain ymples was measured by counting with Insta-Gel (Packard Instrument Co., St. Louis, MO, U S A ) in a Packard liquid scintillation counter. An external standard was used for correction of the quench. The radioactivity of the glucose. lactate, and amino acids was measured by passing the emuent from the column through a flow cell. packed with anthracene, in a liquid scintillation counter (TYCE,1971). Calculations. Brain glucose values were corrected for residual blood glucose in brain (3"" of the fresh wt) as described by FLOCKer a/. (1969). The flux of 14C from glucose in plasma to metabolites in the brain was caleulated by expressing I4C in a metabolite as a function of the specific activity of the glucose in plasma and thus obtaining its plasma glucose equivalent. Similar calculations were done to obtain brain glucose equivalents and brain glutamate equivalents in metabolites. The statistical significance of the results was determined by Student's f test. Data are given as means f S.F.M. RESULTS
Changes of hehacior of the rats after M A 0 1 and 5-HTP
A single injection of 5 - H T P (30mg/kg, i.v.) produced n o abnormal behavior in the rats. However. the combined treatment of Lilly-51641 (10 mg/kg. i.p.) and 5-HTP (30 mg/kg, i.v.) resulted in ataxia, flexion, stretch of limbs and body, intensive tremors, and heavy salivation. T h e dark red color of the blood from such animals suggested a lack of oxygen. When the rats were treated with a lower dose of 5-HTP (10mg/kg, i.v.) after the Lilly-51641 (10 mg/kg, i.p.), only slight involuntary movement of the toes and light salivation were seen. This abnormal behavior occurred about S m i n after the injection of 5-HTP and returned t o normal after about 3 W 5 min. Changes of 5-HTP and 5-HT in brain T h e maximum concentrations of 5 - H T P and 5-HT were found in the brain at 15 rnin after the injection
6t
Time,
rnin
FIG. 1. Etfects of 5-HTP alone ( e - 4 ) or Lilly-51641 on concentration of 5-HTP in rat plus 5-HTP (D-M) brain. In Figs. 1 and 2, 5-HTP was given to rats at time zero and LilIy-51641 was given at 1 h before 5-HTP. Data shown are mean f S.E. of six determinations.
61 5
5-Hydroxyindoles and cerebral glucose metabolism
I
15
30
I
1
1
45
60
I20
Time,
FIG. 2. Effects of 5-HTP alonc (o---+)
min
or Lilly-51641 plus 5-HTP (m5-HT in rat brain.
~
W)
on concentration of
of 5-HTP when it was injected alonc or with the M A 0 inhibitor (Figs. 1 and 2). Consequently, studies of the effects of 5-hydroxyindoles on ['4C]glucose metabolism were done at this time. When Lilly-51641 was given alone, the concentrations of 5-HTP and 5-HT in the brain were not changed.
of threonine in the brain of rats treated with 5-HTP or with Lilly-51641 but not in the rats that received the combined injection. However, these changes were very minor and were within the range of variation found in untreated rats in this laboratory (TYCE, 1976).
Ckarig~soJ' plasrna aid brain glucose
Radioactivity in brain at 5 rnin after adriiinisrratiori of labeled glucose
5-HTP injection alonc failed to cause a significant increase in the plasma concentration of glucose in rats (Table 1). A small increase in plasma glucose levels occurred in rats receiving only Lilly-51641, but the hyperglycemia was more pronounced after the combined administration of Lilly-51641 and 5-HT (Table 1). Brain glucose was slightly, but not significantly, increased by the injection of 5-HTP alone and was significantly increased by the injection of Lilly-51641. A highly significant increase of brain glucose was found in the rats injected with Lilly-51641 plus 5-HTP (Table 1). Changes of amino acids in brain Changes in the concentrations of amino acids in the brain were not great after the treatments with Lilly-51641 and 5-HTP. Alanine and GABA were significantly increased in the brain of rats treated with 5-HTP alone or with Lilly-51641 and 5-HTP (Table 2). Small increases were found in the concentrations
Compared with the controls. 5-HTP injection caused pronounced increases in the radioactivity as glucose and reduced the amounts of radioacti\e amino acids and of lactate (Table 3). More extensive changes of these proportions were caused by Lilly-51641. When both Lilly-51641 and 5-HTP were injected into the animals, radioactive glucose was increased more than 60';/, and the radioactivity as amino acids was significantly decreased (Table 3). Treatment with 5-HTP changed slightly the specific radioactivity of glucose in the brain 5 min after the injection of labeled glucose (Table 4). The administration of Lilly-51641 also significantly reduced the specific radioactivity of glucose, and a more extensive reduction was found after the combined injection of Lilly-51641 and 5-HTP (Table 4). Similarly, specific radioactivities of the major amino acids in brain were also decreased after the injection of 5-HTP or Lilly-51641 or both (Table 4).
TABLE1. CONCENTRATION OF PLASMA
AND BRAIN GLUCOSE*
Treatment
Plasma Glc (pmol/ml)
Brain glucose @mol/g)
Control ( 6 ) 5-HTP (5) Control (6)
9.85 f 0.35 11.86 f 0.98
2.31 f 0.18
M A 0 1 (6) M A 0 1 + 5-HTP (6)
11.53 _+ 1.02t 17.26 f 1.281
8.90 f 0.42
2.02 f 0.11 1.89 f 0.15 2.54 _+ 0.22t 3.76 f 0.21:
* Figures in parentheses represent the number of animals; values are means f appropriate controls. t P < 0.0s. 1P < 0.001.
S.E.M.
against
KAR-LITWONCand G. M. TYCE
616
TABLF2. CONCENTRATION OF AMINO Substance Taurine Aspartate Threonine Glutamine Glutamate Glycine Alanine GABA
ACIIIS IN B R A I N
(pmol/g*)
Control (6)
5-HTP (5)
Control (6)
MA01 (6)
MA01 + 5-HTP (6)
4.68 f 0.25 3.08 f 0.17 0.47 f 0.02 6.38 f 0.22 11.77 f 0.42 0.62 k 0.02 0.32 f 0.01 1.45 f 0.05
4.19 f 0.47 3.11 f 0.16 0.54 k 0.02t 6.76 k 0.15 11.78 f 0.14 0.67 f 0.03 0.35 f 0.0lt 1.73 k 0.081
4.98 f 0.31 3.14 f 0.12 0.49 f 0.02 6.36 f 0.30 11.32 f 0.21 0.63 f 0.03 0.34 5 0.01 1.61 f 0.11
4.60 f 0.22 3.29 f 0.09 0.56 f 0.02t 6.67 f 0.17 11.42 f 0.33 0.60 f 0.02 0.34 f 0.01 1.63 f 0.10
4.89 f 0.09 3.26 f 0.10 0.54 f 0.02 7.10 f 0.28 11.00 f 0.17 0.70 f 0.04 0.43 f 0.02: 1.99 f O.12t
* Figures in parentheses represent number of animals; values are means f t P < 0.05.
against appropriate controls
S.F.M.
:P < 0.01.
TABLF3. RADIOACTIVITY I N BRAIN 5 min Substance Glucose Lactate Aspartate Glutamine Glutamate Alanine GA BA
AFTER INJECTION or L A B ~ L E DGLUCOSE*
Control (6)
5-HTP (5)
Control (6)
MA01 (6)
MA01 + 5-HTP (6)
42.75 f 2.82 8.31 f 0.79 5.26 f 0.25 4.84 f 0.39 24.48 f 1.40 1.44 k 0.14 2.12 f 0.18
50.63 f 2.54 7.86 _+ 0.68 3.74 f 0.35t 4.06 f 0.32 20.19 f 1.198 1.29 f 0.12 1.68 & 0.16
41.92 f 2.04 7.76 f 0.40 5.34 f 0.04 4.80 f 0.21 24.25 f 1.29 1.51 f 0.10 2.04 f 0.15
53.32 f 3.44t 6.42 f 0.465 3.89 f 0.38t 3.06 f 0.39t 18.33 f 1.665 1.14 f 0.18 1.90 f 0.29
68.24 f 2.12: 6.08 f 0.905 2.02 f 0.13: 2.04 f 0.19: 10.74 f 0.68: 0.78 f 0.13t 1.03 f 0.17:
* Figures in parentheses represent the number of animals; data are expressed as percent of total 14C in brain; values are means f S.E.M. against appropriate controls. t P < 0.01.
:P < 0.001.
$P
< 0.05.
RADIOACTIVITY TABLE4. SPECIFIC
Substance Glucose Aspartate Glutamine Glutamate Alanine GABA
OF G L U C O S ~AND AMINO ACIDS I N RAT BRAIN
(d.p.m./pmol x lo-’)*
Control (6)
5-HTP (5)
Control (6)
MA01 (6)
MA01 + 5-HTP (6)
65.83 f 7.49 5.31 k 0.56 2.37 f 0.36 6.45 f 0.79 14.31 f 2.07 4.51 f 0.61
52.50 f 2.28 2.87 f 0.263 1.42 f 0.Oq 4.06 f 0.18t 8.69 k 0.53t 2.34 f 0.29t
59.46 f 5.25 4.70 f 0.27 2.07 k 0.08 5.91 f 0.40 12.03 f 1.08 3.64 f 0.53
45.26 f 2.861. 2.55 & 0.316 1.00 f 0.165 3.45 f 0.393 7.07 f 1.21t 2.51 f 0.45
37.34 & 2.131 1.29 & 0.145 0.60 f 0.076 2.00 f 0.176 3.78 f 0.66s 1.02 f 0.11s:
* Figures in parentheses represent the number of animals; values are means f t P < 0.05.
S.E.M.
against appropriate controls.
:P < 0.01.
5 P < 0.001.
Incorporation of labeled glucose into its nietabolites in brain
The administration of Lilly-51641 alone or with 5-HTP reduced the total 14C in brain 5 min after the intravenous injection of labeled glucose (Table 5). However, the plasma glucose equivalent in brain was unchanged after treatment with Lilly-51641, with 5-HTP, or with Lilly-51641 plus 5-HTP. This suggested that the uptake of glucose into the brain from the circulation was not increased after these treatments, even though the plasma concentration of glucose was increased (Table 1). The flux of 14C from plasma glucose to lactate and alanine was not appreciably affected by the adminis-
tration of Lilly-51641 or 5-HTP or both (Table 5). O n the other hand, the flux of 14C to the major amino acids other than alanine in the brain was sharply decreased after injection of 5-HTP or Lilly-51641, and the decrease was more pronounced after combined treatment with Lilly-51641 and 5-HTP (Table 5). Equivalents of glucose and glutarnate in their rnetaholites
The brain glucose equivalents in glutamate and in aspartate in the brain were reduced after injection of 5-HTP (Table 6). The injection of Lilly-51641 alone also caused a reduction of brain glucose equivalents
5-Hydroxyindoles and cerebral glucose metabolism TABLE 5. FLUXOF I4C FROM
PLASMA GLUCOSE TO METABOLITES IN BRAIN
Measurement
5 min
617
AFTER THE INJECTION OF LABELED
GLUCOSE*
Control (6)
5-HTP (5)
Control (6)
MA01 (6)
MA01 + 5-HTP (6)
57.63 f 0.35
47.86 f 1.717
56.54 f 3.24
41.66 f 3.83t
36.89 f 1.703
1. Specific activity of
plasma glucose (d.p.m./pnol x 2. Total I4C in brain (d.p.m./g x 3. Plasma glucose equivalent in brain 4. For lactate and alanine 0,; of total Plasma glucose equivalent 5. For amino acids except alanine :,;of total l4C Plasma glucose equivalent
310.23 f 32.81
237.88 f 6.46
275.36 f 10.69 212.56 f 8.293
204.27 f 10.873
5.33 f 0.36
5.00 f 0.23
4.92 i. 0.23
5.24 f 0.30
5.55 f 0.22
9.75 f 0.89 0.52 f 0.07
9.15 f 0.61 0.46 f 0.03
9.27 f 0.43 0.45 f 0.02
7.56 f 0.55 0.39 f 0.03
6.86 f 1.03 0.37 f 0.04
36.70 f 2.06 1.95 f 0.16
29.67 f 1.88 1.47 f 0.06t
36.34 f 1.72 1.78 f 0.06
27.19 f 2.36 1.39 0.06:
15.82 f 1.01 0.87 f 0.041
* Figures in parentheses represent the number of animals; values are means f
S.E.M.
against appropriate controls.
t P < 0.05. 1 P < 0.001
TABLE6. BRAINGLUCOSE
Metabolites Lactate Aspartate Glutamine Glutamate Alanine GABA
EQUIVALENTS I N GLUCOSE METABOLITES I N BRAIN
Control ( 6 )
5-HTP (5)
Control (6)
MA01 (6)
0.39 f 0.04 0.25 f 0.01 0.23 f 0.02 1.16 f 0.08 0.07 f 0.007 0.10 f 0.01
0.36 f 0.04 0.17 f 0.0lt 0.18 f 0.02 0.92 f 0.06$ 0.06 f 0.003 0.08 f 0.005
0.37 f 0.03 0.25 f 0.01 0.23 f 0.01 1.14 f 0.05 0.07 f 0.003 0.10 f 0.01
0.30 f 0.01 0.18 f 0.02t 0.14 f 0.02t 0.86 f 0.07t 0.05 f 0.01 0.09 f 0.01
* Figures in parentheses represent the number
(jtmol/g)* MA01 + 5-HTP (6) 0.33 f 0.04 0.11 f 0.01: 0.11 f 0.01: 0.59 f 0.03: 0.04 f 0.006t 0.055 f 0.008t
of animals; values are means f S.E.M.against appropriate controls.
t P < 0.01. $ P < 0.001. $ P < 0.05.
TABLE7. GLUTAMATE EQUIVALENTS I N
Metabolites
METABOLITES I N BRAIN
(pmol/g)*
Control (6)
5-HTP (5)
Control (6)
MA01 (6)
MA01 + 5-HTP (6)
2.30 f 0.10 2.56 0.18 0.69 f 0.45 1.01 0.04
2.36 f 0.07 2.17 f 0.15 0.76 f 0.07 0.98 f 0.08
2.25 f 0.10 2.50 0.03 0.70 f 0.03 0.96 f 0.06
1.88 0.12t 2.43 f 0.15 0.71 f 0.09 1.21 2 0.20
2.10 f 0.20 2.07 f 0.08t 0.81 f 0.13 1.04 0.13
~
Glutamine Aspartate Alanine GABA
* Figures in parentheses represent the number of animals; values are means f
S.E.M.
against appropriate controls.
t P < 0.05.
in aspartate, glutamine, and glutamate. The administration of 5-HTP and the MAOX significantly decreased the brain glucose equivalent in all major amino acids (Table 6). However, the glucose equivalent in lactate in the brain was not affected by any of the above treatments (Table 6). The brain glutamate equivalents in its metabolites were little influenced by the injection of either 5-HTP or Lilly-51641 or by combined administration (Table 7). The only significant changes from normal were a reduction of glutamine and aspartate after administration of Lilly-51641 and Lilly-51641 plus 5-HTP, respectively.
DISCUSSION Intravenous injection of 5-HTP in our experiments resulted in about a 3-fold higher level of 5-HT in the brain than in controls, and at the same time the level of 5-HTP was also increased. These results coincide with previous findings, for levels of 5-HT in the brain or in peripheral tissue have long been noted to be increased after the administration of its precursors tryptophan or 5-HTP (UDENFRIEND ef al., 1957; KNOTT& CURZON, 1974). JOHNSTON (1968) suggested the presence of two types of M A 0 in rat brain (types A and B), and sero-
618
WONG and G. M. Tvct KAR-LIT
tonin was shown to be metabolized by the type A the plasma glucose equivalent is not increased in enzyme (FULLER,1972; NEFFet al., 1974). Although these experiments under conditions in which concenconvincing proof of the existence of several forms of trations of glucose in plasma were increased, it seems M A 0 has not yet been obtained (JAIN,1977), the in- probable that uptake of glucose by the brain was in hibitor LiIIy-51641 has been shown to be selective some way impaired by treatment with 5-HTP and in its effect of blocking the metabolism of 5-HT by with Lilly-51641. M A 0 (FULLER,1972). The present treatment with Generally, injections of Lilly-51641 and 5-HTP, Lilly-51641 plus 5-HTP resulted in an approx 4-fold singly or together, did not change the amino acid higher level of brain 5-HT than that in the intact concentrations in the brain in the present studies. rats. 5-HTP was also increased after the combined 5-HTP caused a modest increase in the levels of administration of Lilly-51641 and 5-HTP. The glu- alanine and GABA. Alanine and GABA levels were cose metabolism under the influence of the MA01 also increased by 5-HTP injection after the and of 5-HTP was investigated at times when 5-HT Lilly-51641 treatment, but Lilly-51641 given alone did or 5-HTP was at its highest level after the treatment. not alter their levels. In the present experiments, administration of Changes in 5-HT levels in brain have been shown to be associated with pronounced behavioral changes 5-HTP and of Lilly-51641 reduced the conversion of in animals. Tremors have also been shown to occur glucose to GABA and other major amino acids in et al., 1971; the brain. This reduced metabolism of glucose was in mice by other investigators (MANNISTO MODIGH,1974),and it is suggested that these manifes- probably in part the reason for its accumulation in tations were due to large amounts of 5-HT acting brain. However, it is of interest that although the flux on central 5-HT receptors (ALGERI& CERLETTI, 1974; to GABA from glucose was decreased, the concenJACOBY et al., 1976). tration of GABA increased both after 5-HTP treatBoth hypoglycemic and hyperglycemic effects have ment and after the combined treatment. It has been been reported previously after the administration of suggested (POPOV& MATTHIES, 1969) that MA01 can 5-HTP or 5-HT to animals. Variations in the glyce- increase the GABA level by an inhibition of GABA mic response to these agents may be related to transaminase (EC 2.6.1.19), but in our experiments the species; thus, dogs (SIREKet al., 1966) and rabbits administration of the MA01 alone did not signifiet a[.,1952) usually show hyperglycemic re- cantly affect the concentration of GABA. (CORRELL sponses, mice demonstrate hypoglycemia (DARWISH 5-HT can produce major changes in the vasculature & FURMAN, 1975), and rats both hyperglycemia (COR- (SWANK& HISSEN,1964; DESHMUKH & HARPER, RELL et a/., 1952; LEVINE et a/.,1964) and hypoglyce- 1973: RAPELA& MARTIN,1975; MENDELOW et al., mia (MIRSKY et al.. 1957; KOBAYASHI et al., 1960). 19771, and after injection of 5-HTP, increased The responses to 5-HTP and 5-HT in rats may amounts of 5-HT can be expected in the plasma et al., UDENFRIEND depend on the strain; thus, Wistar (KOBAYASHI et al., 1957). Thus, part of the decreased 1960) and Carworth (MIRSKY el al., 1957) rats usually flux of I4C from glucose in the plasma to its metaboet lites in the brain observed after the injections of show hypoglycemia, and Sprague-Dawley (LEVINE a/., 1964) and albino (CORRELL et a/., 1952) rats 5-HTP (Table 5) could be attributed to a decrease demonstrate hyperglycemia. Another factor in rats is in cerebral blood flow (DESHMUKH & HARPER,1973) undoubtedly the state of nutrition of the animals; the induced by 5-HT present in the plasma. However, hypoglycemic response usually occurs in fasted ani- the conversion of I4C from brain glucose to metabomals (MIRSKYet a!., 1957; KOBAYASHI et al., 1960). lites in the brain was also inhibited and to a similar et a!. (1971) have also shown that extent by the 5-HTP treatments (Table 6). Because LUNDQUIST another MAOI, N-methyl-N-2-propynylbenzylaminethe glucose in the brain had been corrected for glu(pargyline), can cause hypoglycemia in mice when cose in the residual blood in the brain (although not given alone or in combination with 5-HTP. On the for glucose in the cerebrospinal flaid), this decreased other hand, the injection of 5-HT and N-benzyl- conversion of brain glucose to its metabolites suggests p-(isonicotinoy1hydrazino)propionamide (nialamide) that there is. in fact. an inhibition of glucose metabolresulted in hyperglycemia in mice (DARWISH & FUR- ism in the brain after the injections of 5-HTP, MAN, 1974). However, their effects may be mediated although an effect on cerebral blood flow cannot be by changes in the concentrations of other amines, excluded. such as tyramine (FULLER,1972), rather than 5-HT. Although the flux of 14C from glucose to the amino Glucose passes the blood-brain barrier by a special acids in brain was reduced by treatment with 5-HTP transport mechanism (BETZet al., 1976), and the in- or with Lilly-51641, the flux to lactate and alanine flux of glucose from the circulation to the brain is appeared to be unaffected. This suggests that a point influenced and regulated by the concentration of glu- of inhibition was at the entry of pyruvic acid into cose in plasma (PRATT,1976). Although the plasma the tricarboxylic acid cycle. A similar decreased flux of carbon from glucose glucose equivalent, as calculated in these experiments, is not a precise measurement of glucose uptake by to amino acids in the brain was observed previously the brain from plasma (TYCE, 1971)-some 14C is lost after DOPA administration (TYCE& OWEN,1973; as C0,-it does provide an index of uptake. Since TYCE,1976) and after phenylalanine or tryptophan
5-Hydroxyindoles and cerebral glucose metabolism administration (BARBATO & BARBATO,1969). This suggests a common effect of aromatic amino acids on glucose metabolism in brain. This effect would be caused by a competition by the aromatic amino acids for cofactors, especially pyridoxal-5-phosphate, that are necessary for the formation of glutamate, glutamine, aspartate, and GABA from tricarboxylic acid cycle intermediates. Pyridoxal-5-phosphate is also a cofactor for the enzyme L-aromatic amino acid decarboxylase and for tyrosine aminotransferase (EC 2.6.1.5) (NICKLAS& BERL,1973). It would be of interest to determine whether the metabolism of glucose is inhibited to a similar extent in rats fed with pyridoxine-deficient diets or by the administration of drugs that bind pyridoxal phosphate (for example, penicillamine and isoniazid). These problems are currently being investigated in our laboratory. However, in the present experiments, the inhibition of flux of carbon from labeled glucose to amino acids was greatest in rats in which the amine (rats treated with LilIy-51641 plus 5-HTP) rather than the amino acid precursor (rats treated with 5-HTP) was increased. Further experiments are needed to determine whether the intraventricular injection of the amines 5-HT and dopamine would elicit similar inhibition. It would also be important to ascertain what effect the injection of an inhibitor of central and peripheral decarboxylatjon (for example, 4-bromo-3-hydroxybenzyloxyamine. NSD 1055) has on glucose utilization of the brain. Compared with previous reports from our laboratory (TYCE, 1976) and from other institutions (SHIMADA er al., 1970) in which liquid nitrogen was used to kill rats or mice, the use of the brain-blowing technique to fix brain tissue showed a higher percentage of radioactivity as glucose and amino acids and a markedly lower amount as lactate. These data undoubtedly reflect a minimizing of postmortem artifacts when the more rapid method of killing was used. AcknowledgementsThe authors are greatly indebted to Mrs. JAUNEITA OGG and Mr. CURTIS GRABAUfor their skillful technical assistance.
619
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